An axonemal intron splicing program sustains Plasmodium male development

Differentiation of male gametocytes into flagellated fertile male gametes relies on the assembly of axoneme, a major component of male development for mosquito transmission of the malaria parasite. RNA-binding protein (RBP)-mediated post-transcriptional regulation of mRNA plays important roles in eukaryotic sexual development, including the development of female Plasmodium. However, the role of RBP in defining the Plasmodium male transcriptome and its function in male gametogenesis remains incompletely understood. Here, we performed genome-wide screening for gender-specific RBPs and identified an undescribed male-specific RBP gene Rbpm1 in the Plasmodium. RBPm1 is localized in the nucleus of male gametocytes. RBPm1-deficient parasites fail to assemble the axoneme for male gametogenesis and thus mosquito transmission. RBPm1 interacts with the spliceosome E complex and regulates the splicing initiation of certain introns in a group of 26 axonemal genes. RBPm1 deficiency results in intron retention and protein loss of these axonemal genes. Intron deletion restores axonemal protein expression and partially rectifies axonemal defects in RBPm1-null gametocytes. Further splicing assays in both reporter and endogenous genes exhibit stringent recognition of the axonemal introns by RBPm1. The splicing activator RBPm1 and its target introns constitute an axonemal intron splicing program in the post-transcriptional regulation essential for Plasmodium male development.


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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.The sample size was determined based on similar experiments previously published in the lab, including "EB1 decoration of microtubule lattice facilitates spindle-kinetochore lateral attachment in Plasmodium male gametogenesis" (Nature Communications, 2023); "Apical anchorage and stabilization of subpellicular microtubules by apical polar ring ensures Plasmodium ookinete infection in mosquito" (Nature Communications, 2022); "A malaria parasite phospholipid flippase safeguards midgut traversal of ookinetes for mosquito transmission" (Science Advances, 2021).The sample sizes were considered sufficient to conduct the experiments with adequate statistical power using t-tests and other tests.
In the bioinformatic analysis of global intron retention, low-expressed genes (TPM below 30) were excluded.To eliminate potential false positives, introns with peak scores exceeding 50% of adjacent exons were discarded in parental parasites.In mutant parasites, introns with peak scores below 50% of neighboring exons were also omitted.
All measurements were replicated biologically.The number of biological replicates is stated in each figure legend.All attempts at replication were successful.The mosquito infection experiment was performed once for some modified (gene tagging) parasites in Supplementary Table 1, which is sufficient to confirm the normal life cycle progression for these parasites.
For the parasite infection assay, mice and mosquitoes were randomly divided into corresponding groups.
In this study, there were not any experiment which required the blinding of the samples.
The study did not use wild animals.
Female mice were used for the Plasmodium yoelii parasite infection.Up to five mice per cage were arranged, which make it easier and more cost-effective to maintain.For mosquito infection via mouse blood feeding, only female mosquitoes were used.
No field-collected samples were used in this study.